Pyrrolizidine Alkaloids as Hazardous Toxins in Natural Products: Current Analytical Methods and Latest Legal Regulations

Pyrrolizidine alkaloids (PAs) are toxic compounds that occur naturally in certain plants, however, there are many secondary pathways causing PA contamination of other plants, including medicinal herbs and plant-based food products, which pose a risk of human intoxication. It is proven that chronic exposure to PAs causes serious adverse health consequences resulting from their cytotoxicity and genotoxicity. This review briefly presents PA occurrence, structures, chemistry, and toxicity, as well as a set of analytical methods. Recently developed sensitive electrochemical and chromatographic methods for the determination of PAs in honey, teas, herbs, and spices were summarized. The main strategies for improving the analytical efficiency of PA determination are related to the use of mass spectrometric (MS) detection; therefore, this review focuses on advances in MS-based methods. Raising awareness of the potential health risks associated with the presence of PAs in food and herbal medicines requires ongoing research in this area, including the development of sensitive methods for PA determination and rigorous legal regulations of PA intake from herbal products. The maximum levels of PAs in certain products are regulated by the European Commission; however, the precise knowledge about which products contain trace but significant amounts of these alkaloids is still insufficient.


Introduction
Natural products have been used for centuries in traditional medicine to treat and prevent diseases.Nowadays, many drugs are obtained from plant raw material, especially from medicinal herbs, and natural substances isolated from medicinal plants are considered candidates for new drugs [1].Moreover, other natural sources, including fungi, lichens, bacteria, and marine organisms, also provide valuable material for the pharmaceutical industry.On the other hand, due to the growing interest in healthy lifestyles, the use of supplements and medicinal products of plant origin is becoming popular among consumers.The market for foods classified as dietary supplements is developing dynamically.There is a misconception that these products are always safe and side-effect free.Importantly, these products are consumed without proper medical supervision.To make dietary supplements safe for consumers, relevant legal regulations and market control are needed.
This review provides information on the occurrence of PAs in the environment along with insight into secondary pathways of PA contamination of plant products.It focuses on analytical techniques applied for PA determination in medicinal and food products of plant origin.Special emphasis is put on liquid chromatography (LC) methods combined with mass spectrometry (MS) detection.However, other methods are discussed as well.Recently published reviews [25,[27][28][29][41][42][43] summarize methods for determining PA, mainly methods with MS detection using various types of analyzers, which are characterized by different levels of sensitivity.The most modern and sensitive type of detector is currently Orbitrap, and there are relatively few reports on methods using this type of analyzer in other reviews.This review presents PA testing methods using both basic types of analyzers and the most modern ones, based on the latest literature reports.This review also provides much more detailed information on the mechanisms of PA-induced cytotoxicity and genotoxicity, as well as their LD 50 values, which are not included in existing reviews.It also provides a broad summary of European regulations regarding these dangerous pollutants.

Natural Occurrence of PAs, Health Risks, and Possible Routes of Human Intoxication
In general, alkaloids are a diverse class of naturally occurring organic compounds.They are characterized primarily by the presence of at least one nitrogen atom in the structure.The inherence of nitrogen in the form of a heteroatom in the rings, in the form of an amino group or, less often, an amide group, causes the basicity of these compounds.This broad group also includes related ones that have neutral or even slightly acidic properties.Alkaloids may also contain other heteroatoms such as sulfur and, less commonly, phosphorus, chlorine, and bromine [44].These compounds are synthesized by a wide range of organisms; they can be found in bacteria, fungi, plants, and animals [45,46].Alkaloids have a wide range of pharmacological activities, such as antimalarial, antiasthmatic, anticancer, cholinomimetic, vasodilatory, antiarrhythmic, analgesic, antibacterial, and antihyperglycemic effects.Due to their strong biological activity, many alkaloids are used in traditional or modern medicine or serve as starting points for drug discovery [47].
Alkaloid synthesis is a secondary metabolic process.The necessary compounds for alkaloid synthesis are pyruvic acid and acetyl-CoA.Figure 1 shows the synthesis of alkaloids in the metabolic system of plants.The synthesis of alkaloids in the plant is a multi-step process.The first stage is photosynthesis, which produces the monosaccharide D-glucose, which is used as a substrate in the next stage of the Krebs cycle.PAs are compounds containing nitrogen in fused heterocyclic rings, produced in the ornithine metabolism pathway (see Figure 1), while L-ornithine is derived from L-glutamate [21].There were hundreds of structurally different PAs discovered.The occurrence of PAs, their propagation, toxicity, and chemistry are described in detail in the next paragraphs.
PAs are synthesized by a wide variety of plant species.They were identified in over 6000 plants [48], in families: Boraginaceae (all genera), Asteraceae (Senecioneae, Eupatorieae), and Fabaceae (Crotalaria), as natural toxins providing protection against animals feeding on plants [23,49].PA-containing plants are common weeds and are considered invasive and harmful to the environment because they may contaminate the raw plant material.On the crop fields, PA-containing plants and their parts or seeds can contaminate soil and get into harvested cereals, herbs, or vegetables.Accidental mixing of PA-containing plants with plants intended for fodder may lead to contamination of prepared feeds and grains that are subsequently eaten by animals.That makes food of animal origin, such as milk and eggs, a health hazard [23,[48][49][50][51]. Bees can ingest PAs containing pollen, and then they produce contaminated honey [52].Therefore, it leads to PA contamination of the whole food chain.
ecules 2024, 29, x FOR PEER REVIEW 3 of There were hundreds of structurally different PAs discovered.The occurrence of PA their propagation, toxicity, and chemistry are described in detail in the next paragraph PAs are synthesized by a wide variety of plant species.They were identified in ov 6000 plants [48], in families: Boraginaceae (all genera), Asteraceae (Senecioneae, Eupatorie and Fabaceae (Crotalaria), as natural toxins providing protection against animals feedi on plants [23,49].PA-containing plants are common weeds and are considered invas and harmful to the environment because they may contaminate the raw plant mater On the crop fields, PA-containing plants and their parts or seeds can contaminate soil a get into harvested cereals, herbs, or vegetables.Accidental mixing of PA-contain The European Food Safety Authority (EFSA) recognized PAs as potential toxic components of feed and food, which can become a significant public health problem due to the high risk of contamination of food of plant or animal origin [9].Possible routes of intoxication with PAs are shown in Figure 2. Generally, PA intoxication is possible through ingestion of PA-containing herbal products or PA-contaminated foods, such as tea, herbs, vegetables, spices, and salads.PA-containing plants are numerous and widespread.Human intoxication can occur through the consumption of contaminated basic food products and some herbal remedies [15].
to the high risk of contamination of food of plant or animal origin [9].Possible routes intoxication with PAs are shown in Figure 2. Generally, PA intoxication is possib through ingestion of PA-containing herbal products or PA-contaminated foods, such tea, herbs, vegetables, spices, and salads.PA-containing plants are numerous and wid spread.Human intoxication can occur through the consumption of contaminated ba food products and some herbal remedies [15].

Chemistry of PAs
So far, more than 500 PAs have been found and their structures determined [5 Taking into account the form of N-oxides, over 900 structures are known.PAs are het ocyclic compounds, they include a group of basic ester compounds (mono-and diester which structurally include a combination of amino alcohols with mono-or dicarboxy acids [22].They are pyrrolizidine or necine derivatives, esters, and diesters [21].P undergo acidic or basic hydrolysis, giving basic necine-type amino alcohols that can assigned as PA groups according to the necine base: otonecine, retronecine, heliotridin and platynecine.The biosynthesis of PAs begins with the amino acid ornithine, whi leads to the generation of putrescine and then spermidine.One molecule of putresci and spermidine is transformed into homospermidine [54].Homospermidine is deam nated and creates the necine base skeleton, esterified with a necic acid [55].The neci base forms esters with small organic acids and generates cyclic PAs (retronecine a otonecine type) and open-ringed PAs (heliotridine type) (Figure 3).In detail, a molecu of PAs consists of the core structure-pyrrolizidine, a bicyclic aliphatic hydrocarb consisting of two fused five-membered rings with a nitrogen atom between them and many structures with a double bond in the 1,2 positions (sub-group of PA 1,2-unsaturated).The main toxic effects of PAs are on the liver and lungs.1,2-unsaturat PAs are genotoxic and cause liver cancer in experimental animals.The Scientific Panel

Chemistry of PAs
So far, more than 500 PAs have been found and their structures determined [53].Taking into account the form of N-oxides, over 900 structures are known.PAs are heterocyclic compounds, they include a group of basic ester compounds (mono-and diesters), which structurally include a combination of amino alcohols with mono-or dicarboxylic acids [22].They are pyrrolizidine or necine derivatives, esters, and diesters [21].PAs undergo acidic or basic hydrolysis, giving basic necine-type amino alcohols that can be assigned as PA groups according to the necine base: otonecine, retronecine, heliotridine, and platynecine.The biosynthesis of PAs begins with the amino acid ornithine, which leads to the generation of putrescine and then spermidine.One molecule of putrescine and spermidine is transformed into homospermidine [54].Homospermidine is deaminated and creates the necine base skeleton, esterified with a necic acid [55].The necine base forms esters with small organic acids and generates cyclic PAs (retronecine and otonecine type) and open-ringed PAs (heliotridine type) (Figure 3).In detail, a molecule of PAs consists of the core structure-pyrrolizidine, a bicyclic aliphatic hydrocarbon consisting of two fused five-membered rings with a nitrogen atom between them and in many structures with a double bond in the 1,2 positions (sub-group of PAs, 1,2-unsaturated).The main toxic effects of PAs are on the liver and lungs.1,2-unsaturated PAs are genotoxic and cause liver cancer in experimental animals.The Scientific Panel on Contaminants in the Food Chain (CONTAM Panel) of the European Food Safety Authority published a scientific opinion on the risks to public health related to the presence of pyrrolizidine alkaloids in food and feed.The CONTAM Panel concluded that only 1,2-unsaturated pyrrolizidine alkaloids are toxic and may act as genotoxic carcinogens in humans [9,56].The necine base is often retronecine, heliotridine, or otonecine.Necic acids are varied organic acids, when they are dicarboxylic, they form macrocyclic PAs.PAs demonstrate great structural diversity.With the large number of necic acids, which can be combined with a set of necine bases, a huge structural diversity of PAs is possible [22].Moreover, modifications including N-oxidation of the tertiary nitrogen of the necine base, hydroxylation of the necine base and the necic acid, and acetylation of hydroxy groups further enhance these possibilities.Chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915 [57] are presented in Table 1.The chemical structures of PAs, which should also be monitored in food and feed, according to the EFSA opinion [8,9], are presented in Table 2. N-oxidation is a special type of modification because it is reversible.N-oxidation of the tertiary amine nitrogen significantly changes the properties of a native PA.The main point is that PANOs become more polar and highly water soluble.In plants, what is most important is that the major fraction of PAs is present as PANOs [22].
are varied organic acids, when they are dicarboxylic, they form macrocyclic PAs.PA demonstrate great structural diversity.With the large number of necic acids, which c be combined with a set of necine bases, a huge structural diversity of PAs is possible [2 Moreover, modifications including N-oxidation of the tertiary nitrogen of the necine bas hydroxylation of the necine base and the necic acid, and acetylation of hydroxy grou further enhance these possibilities.Chemical structures of PAs listed in Annex 1 to t Commission Regulation (EU) 2023/915 [57] are presented in Table 1.The chemical stru tures of PAs, which should also be monitored in food and feed, according to the EFS opinion [8,9], are presented in Table 2. N-oxidation is a special type of modification b cause it is reversible.N-oxidation of the tertiary amine nitrogen significantly changes t properties of a native PA.The main point is that PANOs become more polar and high water soluble.In plants, what is most important is that the major fraction of PAs is pr sent as PANOs [22].
The determination of PANO concentration is necessary because, in vivo, the N-oxides can be biotransformed into the corresponding PA-free bases.This process tak place after ingestion in the gastrointestinal tract and in the liver, mediated by, respe tively, the intestinal microbiota and hepatic cytochrome P450 monooxygenases [5 When reduced, N-oxide-derived PAs are subsequently metabolized to the hepatotox pyrrole.Therefore, the total PA content of the tested material may be underestimate unless both PA and PANO contents are determined.

No. Name Alkaloid Chemical Structure Chemical Structure of Corresponding N-Oxide
Possible co-elution of 9 and 10 with, respectively:

9b, 10b
Jaconine, jaconine-N-oxide The determination of PANO concentration is necessary because, in vivo, these N-oxides can be biotransformed into the corresponding PA-free bases.This process takes place after ingestion in the gastrointestinal tract and in the liver, mediated by, respectively, the intestinal microbiota and hepatic cytochrome P450 monooxygenases [58].When reduced, N-oxide-derived PAs are subsequently metabolized to the hepatotoxic pyrrole.Therefore, the total PA content of the tested material may be underestimated unless both PA and PANO contents are determined.

Toxicity of PAs and Regulation Limits
PAs are potentially hepatotoxic compounds that lead to human poisoning through the food chain (such as tea, herbs, botanical preparations, spices, and vegetables).The toxicity of PAs depends on their physical properties and their metabolism in the liver [59].PAs very often produce pyrrolizidine alkaloid N-oxides (PANOs), of lower toxicity, that cannot be directly converted to hydroxypyrrolidines. PANOs generally exhibit low toxicity but undergo toxic processes in vivo and cause toxification through biotransformation to the corresponding PAs [60].PANOs are reduced to free bases in the intestines after their ingestion, which was described further.The hepato-and cytotoxicity mechanisms of PAs are shown in Figure 4.

Toxicity of PAs and Regulation Limits
PAs are potentially hepatotoxic compounds that lead to human poisoning through the food chain (such as tea, herbs, botanical preparations, spices, and vegetables).The toxicity of PAs depends on their physical properties and their metabolism in the liver [59].PAs very often produce pyrrolizidine alkaloid N-oxides (PANOs), of lower toxicity, that cannot be directly converted to hydroxypyrrolidines. PANOs generally exhibit low toxicity but undergo toxic processes in vivo and cause toxification through biotransformation to the corresponding PAs [60].PANOs are reduced to free bases in the intestines after their ingestion, which was described further.The hepato-and cytotoxicity mechanisms of PAs are shown in Figure 4.The oxidation of PAs is a metabolic process that occurs in the liver.This process is crucial as it leads to the formation of reactive PA metabolites [62] (see Figure 4a).During oxidation processes in the liver, a hydroxyl group is attached to the alkaloid molecule and the carbon adjacent to the nitrogen atom.The alkaloid thus formed is unstable and is immediately dehydrated into the dehydro-pyrrolizidine alkaloids (DHPAs).As a result, a second double bond is formed in the necine molecule.At a later stage, after hydrolysis, an aromatic pyrrole moiety is generated (dehydropyrrolizidine-DHP), with the ester groups removed.In the final process, there are formed pyrrole-protein adducts and pyrrole-DNA adducts, which are responsible for cytotoxicity and genotoxicity (see Figure 4b) [61,63].PA-induced liver injury is suspected to be associated with the consumption of PA-containing herbal products, and the pyrrole-DNA adducts were detectable in patients' blood samples [61].
PAs may cause acute toxicity, mutagenicity, chromosomal aberrations, the formation of abnormal cross-links between DNA strands and DNA-protein bonds, and megalocytosis [23,64,65].They are responsible for: the formation of cancer cells [56,66,67], disturbances of the liver metabolism [23,66], liver necrosis [19], fibrosis [19], cirrhosis [19], photosensitization [19,23], diarrhea [19,23], incoordination [8,28], aggressive behavior [8], body weight loss [8,23], loss of appetite [8,23].Poisoning with PAs is usually asymptomatic.By the time symptoms of liver damage appear, the disease process has already developed and caused death in a short time [23,68].The LD 50 values of the most important PAs are known [2].However, the increase or inhibition of cytochrome P450 activity by drugs may also change the toxicity of PAs.PA metabolites react with SH groups located in glutathione or cysteine.Therefore, a diet rich in glutathione, taurine, cysteine, and methionine may reduce the toxicity of ingested PAs [69].PA toxicity also depends on exposure time, dose, and the organism's susceptibility.PAs cause acute toxicity within 1-6 days, while doses of 0.1 mg/kg body weight per day cause chronic toxicity.In humans, the toxic dose ranges from 0.1-10 mg/kg body weight per day [10].Table 3 shows the LD 50 of the more important PAs studied in vivo in animal models [2] and predicted using the computer software TOPKAT (Discovery Studio 2019 (Accelrys, Inc., San Diego, CA, USA) [70].[10].Following a review of the available data, the EMA (HMPC) considered a harmonized approach to implementing appropriate controls for the markets in EU countries.A contamination level of herbal medicinal products leading to a daily intake of a maximum of 1.0 µg PAs per day during a transitional period of 3 years was considered acceptable from a public health point of view.After this period, producers of herbal medicinal products should be required to take the necessary measures to reduce the contamination to a level resulting in a daily intake not exceeding 0.35 µg PAs per day [10,14,71].The report concluded that contamination of herbal products (food or medicines) with PAs is not a new matter, but new, sensitive analytical methods can now detect very low levels of PAs.Tea and herbal teas are the largest contributors to human exposure to pyrrolizidine alkaloids, as well as pollen-based supplements.It was found that the exposure to pyrrolizidine alkaloids associated with the consumption of honey is lower.It has also been found that herbal dietary supplements may contribute significantly to human exposure to PAs, but incidence data are insufficient [10].Generally, the human intake of PAs through food and herbal medicinal products has probably remained constant over the last few years, and statistically, the incidence of liver hemangiosarcoma in humans is very low.EMA has emphasized that once the problem with PA contamination of herbal medicinal products has been identified, regulatory actions to mitigate the problem must be considered.
The European Food Safety Authority (EFSA) prepared in 2016 a report on chronic and acute dietary exposure to PAs in the European population through the consumption of foods of plant origin [11].This scientific report focuses on the 28 PAs selected based on the EFSA scientific opinion from 2011 [9], which identified key PAs in tea and herbal infusions.The 28 selected PAs include echimidine, heliotrine, lycopsamine, intermedine, erucifoline, senecionine, seneciphylline, monocrotaline, jacobine, senecivernine, retrorsine, europine, lasiocarpine, senkirkine, and their N-oxide forms.A total of 274,632 analytical results on PAs in food samples were available, accounting for a total of 19,332 food samples.The number of PAs analyzed per sample ranged between one and 28.To avoid underestimation of the presence of PAs, only those samples with a minimum number of PAs were selected.Special attention was paid to the presence of two additional PAs, riddelliine and riddelliine-N-oxide, due to their toxicity.These two PAs were analyzed in 301 samples of tea and herbal infusions, and in all cases, they were reported below the Limit of Quantification (LOQ).Food samples were mainly tea and herbs for infusion, and honey samples A total of 294 samples of food supplements were also available.In addition to honey samples, 825 food samples of animal origin were also part of this data set, with 97% of them having all analyzed PAs as left-censored data.Previous studies demonstrated that the levels of PAs in animal-derived food are much lower than those that can be found in food commodities such as tea and herbal infusions.Among 746 samples of animal origin, only occasional low levels of PAs in milk samples were found, mostly with single PAs in their free base form.
Except for two egg samples, PAs were absent in the milk products, eggs, meat, and liver samples analyzed.
On 27 July 2017, the EFSA Panel on Contaminants in the Food Chain (CONTAM) published a statement on the risks to human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal teas, and dietary supplements [12].The CONTAM Panel established a new benchmark of 237 µg/kg body weight per day to assess the carcinogenic risks associated with PAs and concluded that exposure to PAs poses a potential risk to human health, especially for people who frequently consume large amounts of tea, herbal teas, and herbal based medicines.The conclusion was also that the younger segment of the population is particularly vulnerable.
In the document of the Standing Committee on Plants, Animals, Food and Feed (PAFF Committee) of 17 April 2018, there is also a reference to PAs [13].According to the Committee, consideration should be given to setting maximum PA levels for the following foods: tea and herbal infusions, tea for babies and small children, herbal dietary supplements derived from plants containing PAs, and dietary supplements accidentally contaminated with plants containing PAs, honey, and pollen-based dietary supplements.
In the document Commission Regulation (EU) 2023/915 dated 25 April 2023 these maximum levels for PAs were maintained [57].Legally, the foodstuffs listed in the Annex to the Regulation placed on the market before 1 July 2022 may be marketed until 31 December 2023.After 1 July 2022, each product covered by the regulation should obligatorily meet the legal requirements in this area.
A European Pharmacopoeia Commission at the European Directorate for the Quality of Medicines (EDQM) published on 1 July 2021 in Supplement 10.6 of the European Pharmacopoeia (Ph.Eur.) the new general chapter "Contaminant pyrrolizidine alkaloids (2.8.26)" [73].This general chapter, which describes 28 target PAs, allows for the use of any procedure consisting of chromatography coupled with MS/MS or high-resolution MS that meets the validation requirements given in the chapter.This approach was adopted because there is considerable variation in the composition and matrices of the herbal drugs, as well as in the applicable limits, making it difficult to describe all the methods suitable for quantitative analysis of the target PAs [73].Performance criteria for method validation for PAs are given in the document Commission Implementing Regulation (EU) 2023/2783 dated 14 December 2023 [74] and in the Guidance Document on Performance Criteria for Methods of Analysis for Mycotoxins and Plant Toxins in Food and Feed from the European Union Reference Laboratory (EURL) [75].
All the regulators indicate that the presence of PAs in food products can be minimized or prevented by the application of good agricultural and harvest practices.The European Tea and Herbal Infusions Industry has developed a Code of Practice to prevent and reduce pyrrolizidine alkaloid contamination in agricultural commodities used in the manufacture of tea and herbal infusions.This is designed to minimize contamination of materials at the primary producer level [77].To prevent and reduce PA contamination, management practices such as effective weed control and careful monitoring of animal feed are crucial.It is also important to note that total eradication of PA-containing plants is not feasible or ecologically desirable [3].

Sample Preparation for the PAs Containing Materials
PA determination is closely related to the appropriate sample preparation.Efficient PA extraction from biological material is required for a valid determination.Analysis of trace amounts of PAs requires proper sample preparation to increase the PA concentration and remove compounds that interfere with the analysis.Various techniques for PA extraction from medicinal plants are described in the literature [24,31,32].The applied sample preparation method should efficiently extract the PAs as well as PANOs at the same time.Therefore, the use of polar organic solvents or aqueous solutions is preferred due to the high polarity of PANOs [15,32].For the PAs and PANOs extraction from different matrices, the most frequently used procedures are solid-liquid extraction (SLE), e.g., sample/methanol [78], and liquid-liquid extraction (LLE), e.g., chloroform/methanol [79].SLE methods are variations based on maceration or percolation, with additional use of other factors such as sonication, high pressure, or solvent modification.Maceration is the process when the herbal material is continuously soaked with solvent, while during percolation, the solvent flows through the plant material.Generally used solvents are dilute aqueous acids: 0.05 M sulfuric acid, 0.15 M hydrochloric acid solution, 0.5% formic acid, and polar organic solvents, e.g., acidified methanol or acetonitrile [31].Organic solvents such as chloroform and dichloromethane may also be used, but this approach is problematic because it requires additional steps.The extract should be dissolved in an aqueous acidic solution and washed with a non-polar solvent, e.g., chloroform, to remove less polar material (such as fats, waxes, and terpenes).The addition of ammonia makes the solution strongly basic, and the PAs are extracted back into an organic solvent.Repetition of this process provides extracts clean enough for GC-MS [32].PANOs are less soluble in relatively non-polar solvents, and to determine the entire profile of PA free bases and N-oxides, in the first step, PA free bases are extracted alone, and in the second step, after reduction of the N-oxides, the total content of PAs is obtained.The proportion of the N-oxides can then be determined by calculating the difference between these two measurements [22,80].
Sample purification is often a necessary step, and solid-phase extraction (SPE) is then used, e.g., solid-phase extraction cartridges (SPE) [81].There are two main types of SPE: strong cation exchange (SCX) SPE and reversed-phase SPE.SCX-SPE is a silicabased benzenesulfonic acid-based filler.Its negatively charged sulfonic acid group has a strong cation exchange capacity, and the benzene ring has a certain hydrophobic retention.SCX extracts positively charged basic compounds, such as amines.Reversed-phase SPE is a slightly selective separation technique.Reversed-phase sorbents, mainly based on octadecylsilane ligands (C18), can retain most molecules with hydrophobic character, making them very useful for extracting analytes that are very diverse in structure within the same sample [82].Mixed-mode sorbents (a combination of reversed-phase and cationexchange interactions) are also used in SPE cartridges.For this purpose, SPE is commonly used for the purification of PAs/PANOs from food samples [31].The technique that is very often used for the determination of PAs/PANOs in food samples is QuEChERS (acronym of "Quick, Easy, Cheap, Effective Rugged and Safe").This procedure assumes the simultaneous extraction and purification of the samples and is suitable for extracting a large number of compounds.This procedure is miniaturized and can be successfully applied to the analysis of 21 PAs/PANOs in oregano, significantly reducing the amount of reagents used by ten times in comparison to the classic methodology [83,84].A broad range of extraction procedures to determine PAs/PANOs in dried plants and food supplements was presented in the review [42].

LC-and GC-Methods for PAs Analysis
Following the successful extraction, sample solutions can be analyzed using chromatographic separation techniques HPLC and GC coupled with various types of detection.
The structural diversity of PAs and PANOs is a challenge for analysts.According to EFSA requirements, the total sum and individual amounts of PAs should be determined in plant material [11].Fast qualitative tests to measure total PA content, instrumental methods to determine the PA profile in samples, and sensitive quantification of these alkaloids are needed.Spectroscopic techniques can be utilized in such studies.However, these methods are not sensitive enough to determine trace amounts of PAs.Ultraviolet-visible spectroscopy (UV-Vis) methods and colorimetry are used for PA detection, rather than for quantitative measurements [31].Nuclear magnetic resonance (NMR) spectroscopy methods were applied to PA analysis for structural identification and identity confirmation of novel PAs [85].The use of immunoassays for the determination of PAs is rare, but the classical enzyme-linked immunosorbent assay (ELISA) can be used for the analysis of PAs [86].The technique is hampered by the presence of some cross-reactivity, preventing easy detection of target PAs.The most popular and useful techniques are chromatographic techniques.Preparative HPLC and TLC can be used for PA isolation [32].TLC, GC, and HPLC methods for PA analysis are also used; however, these methods are suitable for working with materials containing quite a high PA content.A recent review of the analysis of PAs in medicinal plants refers to plants from genera that naturally produce these alkaloids: Tussilago (coltsfoot), Symphytum (comfrey), Senecio, Petasites (butterburs), Lithospermum (gromwell), Heliotropium (bloodstone), Cynoglossum, Borago, Brachyglottis Anchusa, and Alkanna [24].To determine trace amounts of PAs, methods based on mass spectroscopy should be used, which is recommended by the Ph.Eur.[73].

Electrochemical Methods for PAs Analysis
Alkaloid detection methods that use detection techniques such as ELISA, and MS/MS are laborious, time-consuming, expensive, and require the use of complex equipment and staff training.Currently, fast, simple, and accurate methods are being developed to monitor alkaloids in real samples.These benefits are related to the widespread use of electrochemical sensors modified with various materials [87][88][89].Electrochemical detection was found to be favorable because it is easy to use, affordable, rapid, and highly sensitive, and the analysis can be performed on-site [43,90].Various electrochemical techniques can be applied for quantitative analysis, mainly cyclic or stripping voltammetry (CV), differential pulse voltammetry (DPV), and more recently, electrochemical impedance spectroscopy (EIS).Using electrochemical biosensors, researchers can identify specific analytes present in biological samples and convert biochemical signals into electrical signals, simplifying quantification [87][88][89].Electrochemical sensors are also convenient because they can be easily miniaturized, demonstrate excellent sensitivity, and are simple to construct and use.Electrodes are used in detection and are mainly composed of carbonaceous materials such as glassy carbon (GCE), pyrolytic graphite (HOPG), screen-printed carbon electrodes (SPCE), and even still mercury electrodes [91].By modifying the surfaces of these electrodes with various materials, good selectivity can be achieved [92].Improvement of sensitivity and resolution is obtained by using the DPV compared to CV, which is important, espe-cially in detecting traces of alkaloids.EIS is very useful in studying surface processes, kinetics, and mechanisms of alkaloid reactions.Recently, the surfaces of electrodes have been additionally decorated with nanoparticulate materials to increase the sensitivity and selectivity of the sensor.Nanomaterials are selected so that their physical and chemical properties match the detected analyte as much as possible.Particular attention is paid then to their chemical composition, crystal structure, orientation of the crystallographic axis, morphology, and dimensions of the nanoparticles.Inorganic and organic nanomaterials used for electrode modification include carbon and metallic nanoparticles, polymer materials, and others [43,90].Nanomaterials have different shapes, such as nanoflowers, nanowires, nanorods, or nanofibers.Due to their specific surface, high conductivity, and electrocatalytic properties, they are widely used to improve detection limits and specificity.The modified electrodes are useful in testing alkaloids in biological, pharmacological, and agri-food matrices.The determination of alkaloids in biological samples: serum, urine, blood, etc., is crucial in forensics and clinical applications [43,90].It is important to determine alkaloids in human body fluids, especially when alkaloid poisoning is suspected.The analysis must be performed immediately, and the analyte has to be identified at low concentrations [93][94][95].Nanomaterial-modified electrodes are also used for the determination of various alkaloids in pharmaceutical samples because the analytical procedures are simple, fast, and accurate.Trace amounts of various alkaloids can be determined individually or simultaneously.To our knowledge, among the very large number of sensors for the determination of alkaloids, only a few sensors designed specifically for the determination of PAs can be found in the literature.
Erdem and coworkers [96] developed a simple and inexpensive electrochemical test based on a single-use sensor for the quantification of senecionine (SEN) in food.SEN was immobilized on the surface of a pencil graphite electrode.The SEN oxidation signal was used to evaluate the sensor using differential pulse voltammetry (DPV).The selectivity of the sensor was also checked in the presence of other similar PAs such as intermedine, lycopsamine, and heliotrine.The detection limit was 5.45 µg/mL.Electrochemical detection of SEN had high sensitivity and good selectivity.The sensor was also tested by examining its use in flour and herbal tea products.
Yang and coworkers [97] developed a visual, easy-to-use, and cost-effective mesoporous silica-based electrochemiluminescence (MPS-ECL) sensor for point-of-care (POC) testing of PAs.ECL activity was found to depend on the PA structure.The intensity of ECL also varies for different PAs in order: monocrotaline > senecionine N-oxide > retrorsine > senkirkine.The POC sensors had excellent linearity, low detection limits (0.02 µM-0.07 µM), and good recovery, indicating good accuracy and practicality.The portable and low-cost sensor is user-friendly and can be used to test PAs in drugs, food products, and clinical samples, which shows promise in the preliminary assessment of PA-induced health risks.The sensor is repeatable and temperature stable and was used to perform on-site PA screening in milk, tea, herbal medicines, and human serum samples.

LC-MS and GC-MS Methods for PAs Analysis
PA contamination of herbal products is usually at low levels, so sensitive analytical methods based on mass spectrometry (MS), such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), are required for their determination.LC-MS is now the preferred method for the determination of PAs [31,32].LC-MS and LC-MS/MS methods have become the most popular approaches to the identification and quantification of PAs as they combine reliability and high sensitivity with ease of sample preparation.A range of mass spectrometer types can be used, including single quadrupoles (MS), ion traps (IT), triple quadrupoles (QqQ; MS/MS), and time of flight (ToF) instruments.The ionization method is primarily electrospray ionization (ESI).Atmospheric pressure chemical ionization (APCI) can also be applied, but it is less sensitive.High sensitivity is provided by the positive electrospray ionization modes (ESI +) [31].In LC-MS methods, the experiment can be run in single-ion monitoring (SIM) or scan mode.
The detection and quantification of PAs using SIM were successfully performed [98].In LC-MS/MS methods, collision-induced dissociation (CID) of the PA molecular ion provides fragment ions that can be used in selected reaction monitoring (SRM) or multiple reaction monitoring (MRM).Applying MRM detection that uses the transition from the molecular ion to the specific fragments of the molecule, the highest sensitivity and specificity in PA analysis could be obtained [33][34][35][36][37][38][39][40][41].SIM detection on MS/MS also was applied for PAs analysis [99].SRM provided greater sensitivity and selectivity than high-resolution SIM on a single quadrupole [100].High-resolution mass spectrometry (HRMS) is the latest approach to the analysis of complex sample matrices, such as plant-based products.The increased resolution of HRMS instrumentation enables the resolution of isotope distributions and the generation of fragmentation paths.It uses mass analyzers such as ToF and Orbitrap, which have high mass resolving power that can be used to generate high-quality results.Nevertheless, HRMS instrumentation does not replace the standard low-resolution mass spectrometers found in many research laboratories [101].The identification and quantification of 25 PAs and N-oxides using LC-Q-ToF/MS was performed [102].A new type of mass spectrometer, the Orbitrap, has brought a significant change to high-resolution mass spectrometry.PAs in botanical samples and PA environmental degradation products were examined using the Orbitrap MS [103][104][105][106][107]. GC-MS analysis of PAs was performed on a single quadrupole spectrometer and was limited to single PA [108], qualitative analysis [109], and the analysis of volatile PAs [110].
An overview of the methods for PA determination in various food products is given in Table 4.

Safety Ensuring, Prevention, and Market Control
The European Union (EU) has a range of tools to ensure food safety, including the Rapid Alert System for Food and Feed (RASFF).It has been set up for exchanging information between member countries and supports the rapid response of food safety authorities to public health.It is effective at every stage of the food chain.An interactive, searchable online database called RASFF Window provides public access to summary information about the most recently transmitted RASFF notifications and allows searching for information on any notification issued in the past (currently limited to 2020 and later) [121].The database was searched on 28 April 2024, using the keyword "pyrrolizidine", and 136 records were collected in Table 5.The permissible PA level was exceeded in the products mentioned.

Conclusions
The growing interest in plant-based medicinal products and dietary supplements should not be associated with the misconception that these products are inherently safe and free of side effects.PAs are a class of natural toxins that draw significant attention due to their presence in honey and medicinal plants.PAs are proven to be carcinogenic, genotoxic, and hepatotoxic.Metabolic activation of PAs leads to the formation of adducts with DNA, which is considered to be the main cause of the carcinogenic effects of PAs.The toxic nature of PAs poses a potential risk to human health.Human consumption of PAs from food and herbal medicinal products has likely remained stable over recent years, but new, sensitive analytical methods can now detect very low levels of PAs.However, once a problem is identified, regulatory action to mitigate it should be considered.Therefore, the development and validation of sensitive analytical methods, especially those based on LC-MS, are of great importance to ensure consumer safety and improve public health.In this review, we introduced sensitive and selective analytical methods for the determination of PAs in various materials.Reporting new cases of contamination is important to ensure the safety of herbal products.

Future Directions
Despite significant progress in the determination of PAs in various matrices, the structural diversity and chemical properties of PAs present unique challenges to analysts.The continuous development of analytical methods is essential to improving the detection and quantification of PAs.From a future perspective, the determination of PA in plant and bee products should be a mandatory preventive step during production.The development of convenient, fast, and sensitive electrochemical sensors can be an alternative to complex mass spectrometry-based methods.

Figure 1 .
Figure 1.A block scheme of the alkaloid biosynthesis as a secondary metabolism of plants.T pathway of PAs biosynthesis is marked with orange arrows and boxes.Based on [21].The fig was prepared using GIMP 2.8.14 (GNU General Public License) software.

Figure 1 .
Figure 1.A block scheme of the alkaloid biosynthesis as a secondary metabolism of plants.The pathway of PAs biosynthesis is marked with orange arrows and boxes.Based on [21].The figure was prepared using GIMP 2.8.14 (GNU General Public License) software.

Figure 2 .
Figure 2. The possible pathways of human exposure to PAs.The figure was prepared using GIM 2.8.14 (GNU General Public License) software.

Figure 2 .
Figure 2. The possible pathways of human exposure to PAs.The figure was prepared using GIMP 2.8.14 (GNU General Public License) software.

Figure 3 .
Figure 3. Biosynthesis of the most representative examples of PAs.Based on [19,55].The figure w prepared using GIMP 2.8.14 (GNU General Public License) software.

Figure 3 .
Figure 3. Biosynthesis of the most representative examples of PAs.Based on [19,55].The figure was prepared using GIMP 2.8.14 (GNU General Public License) software.

Figure 4 .
Figure 4. Mechanism of metabolic processes of PAs, which leads to hepato-and cytotoxicity (a) and hepato-and genotoxicity (b).Adapted from [61,62].The figure was prepared using GIMP 2.8.14 (GNU General Public License) software.

Figure 4 .
Figure 4. Mechanism of metabolic processes of PAs, which leads to hepato-and cytotoxicity (a) and hepatoand genotoxicity (b).Adapted from [61,62].The figure was prepared using GIMP 2.8.14 (GNU General Public License) software.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.

Table 1 .
The chemical structures of PAs listed in Annex 1 to the Commission Regulation (EU) 2023/915.Twenty-one PAs whose concentration should be monitored (n-numbers) and an additional 14 PAs (na numbers) who are known to co-elute with one or more of the requiring investigation 21 PAs.
Possible co-elution of 15 and 16 with, respectively:

Table 2 .
[8]mical structures of PAs not listed in Table1(nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed[8].

Table 2 .
Chemical structures of PAs not listed in Table1(nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed

Table 2 .
[8]mical structures of PAs not listed in Table1(nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed[8].

Table 2 .
Molecules 2024, 29, x FOR PEER REVIEW Chemical structures of PAs not listed in Table 1 (nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed

Table 2 .
[8]mical structures of PAs not listed in Table1(nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed[8].

Table 2 .
Molecules 2024, 29, x FOR PEER REVIEW Chemical structures of PAs not listed in Table 1 (nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed

Table 2 .
[8]mical structures of PAs not listed in Table1(nb numbers), which, according to the EFSA opinion, should also be monitored in food and feed[8].
Molecules 2024, 29, x FOR PEER REVIEW

Table 3 .
LD 50 values of the most important PAs.

Table 4 .
Analytical separation and detection techniques for the determination of PAs in herbal material.

Table 5 .
Notifications from the RASFF Window database regarding exceedances of PA content in food products on the EU market (accessed on 31 May 2024).